Zoologica Scripta Complete mitochondrial genomes throw light on budding speciation in three Biston species (Lepidoptera, Geometridae) RUI CHENG,DAYONG XUE,ANTHONY GALSWORTHY &HONGXIANG HAN Submitted: 22 December 2015 Cheng, R., Xue, D., Galsworthy, A. & Han, H. (2017). Complete mitochondrial genomes Accepted: 13 March 2016 throw light on budding speciation in three Biston species (Lepidoptera, Geometridae). — doi:10.1111/zsc.12184 Zoologica Scripta, 46,73–84. Biston panterinaria, Biston thibetaria and Biston perclara are very closely related species in the genus Biston, judged on the basis of both morphological and molecular evidence. The distri- bution area and altitudes, and host plants of these three species also show both consistency and differences. However, the exact relationship between the three species is unclear. In this study, we used the ‘distance-based method’, the ‘tree-based method’ and Bayesian phyloge- netics and phylogeography to elucidate the relationship between the three species. Phyloge- netic trees based on mitogenomes, COI+CYTB+16S and three nuDNA genes were constructed. The results of the phylogenetic trees revealed that B. thibetaria and B. perclara were derived from B. panterinaria and render the latter paraphyletic. The budding process of speciation is therefore presumed to be the main factor causing a phylogenetic relationship of this pattern. A host shift from broad-leaved plants living at low altitudes to gymnosperms living at relatively high altitudes provides evidence of budding speciation in these three spe- cies. The divergence time suggests that the budding speciation occurred at approximately 1.38 Ma, which is consistent with the Kunlun-Yellow River Movement. Tectonic move- ments occurring around the Qinghai–Tibet Plateau may be the driver of the budding speci- ation. Corresponding author: Hongxiang Han, Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China. E-mail: [email protected] Rui Cheng, Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China and University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing 100049, China. E-mail: [email protected] Dayong Xue, Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China. E-mail: [email protected] Anthony Galsworthy, The Natural History Museum, Cromwell Road, London SW7 5BD, UK. E-mail: [email protected] Hongxiang Han, Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China. E-mail: [email protected] Introduction 1970; White 1978; Futuyma & Mayer 1980; Coyne & Orr The origin of species is a fundamental issue in evolution 2004; Nosil 2012). Budding speciation is one specific type biology. Most biologists agree that speciation should be of peripatric speciation, which, as a concept, was first pro- seen as the evolution of reproductive isolation between posed by Mayr (1954): he envisaged an initially small colo- populations (Coyne 1994; Coyne & Orr 1998, 2004; Noor nizing population becoming reproductively isolated from 2002). Many kinds of speciation have been described, the wider-ranging parental species. However, the proposal including allopatric speciation, parapatric speciation, peri- of this type of speciation did not at first draw attention. patric speciation and sympatric speciation (Mayr 1942, Later, Wiley & Mayden (1985), Harrison (1991, 1998), ª 2016 Royal Swedish Academy of Sciences, 46, 1, January 2017, pp 73–84 73 Budding speciation in three Biston species R. Cheng et al. Frey (1993) and Rieseberg & Brouillet (1994) elaborated (nuDNA) loci, to test the paraphyly of B. panterinaria and the concept of peripheral isolates. In particular, Harrison its consistency with the hypothesis of budding speciation. If (1991) identified peripheral isolates based on molecular evi- this result is deemed authentic, these three species will dence, with species A embedding within paraphyletic spe- form a good case study for studying budding speciation in cies B. In 2003, Funk & Omland improved the concept of Lepidoptera. budding speciation and listed much evidence and interpre- tation, following which the concept of budding speciation Material and methods began to be widely accepted. Funk & Omland defined bud- Specimen sampling and DNA extraction ding speciation, or peripatric and peripheral isolates, as The sampling sites of B. panterinaria, B. thibetaria and always implying a geographically restricted and mono- B. perclara are shown in Fig. 1. Samples for DNA extrac- phyletic daughter species being embedded within a widely tion were preserved in 100% ethanol and stored at distributed and paraphyletic parental species. Subsequently, À20 °C. DNA was extracted using the DNeasy Tissue kit many examples of budding speciation were discovered, (Qiagen, Beijing, China), and vouchers were deposited at involving liverworts, kelps, flowers, insects, snails, frogs and the Museum of IZCAS (the Institute of Zoology, Chinese shrews (Morse & Farrell 2005; Vanderpoorten & Long Academy of Sciences, Beijing, China). Ten mitogenomes 2006; Chen et al. 2009, 2012; Tellier et al. 2009; Grossen- from four lineages of B. panterinaria (Cheng et al. 2015), bacher et al. 2014; Kruckenhauser et al. 2014). three mitogenomes from B. thibetaria and one mitogenome Biston panterinaria (Bremer & Grey, 1853), Biston thi- from B. perclara were obtained. The mitogenomes were betaria (Oberthur,€ 1886) and Biston perclara (Warren, 1899) amplified as described in Yang et al. (2013). Three mito- have since their original description been regarded as chondrial genes and three nuclear genes were obtained specifically valid members of the genus Biston of the family from total specimens from the above-mentioned three spe- Geometridae, Lepidoptera, and are distributed in east Asia, cies and from ten other Biston species, including the cyto- south China and Taiwan, respectively. Importantly, these chrome c oxidase subunit I (COI), cytochrome b (CYTB), three species are closely related species forming Group III lrRNA (16S), elongation factor 1a (EF-1a), wingless (wg) of the genus based on morphological characters (Jiang et al. and glyceraldehyde-3-phosphate dehydrogenase (GADPH), 2011). In addition, the distributional areas and altitudes, through polymerase chain reaction (PCR) amplification. and host plants of these three species show both consis- PCR conditions were as follows: denaturation at 94 °C for tency and differences. The host plants of B. panterinaria 2 min, followed by 40 cycles at 93 °C for 1 min, annealing are mainly broad-leaved arbours (Yu 2001; Zhang & Li temperature (51 °C for COI,47°C for CYTB,56°C for 2001; Cui 2004; Li et al. 2008) below 1500 m, while the EF-1a,58°C for wg and 55 °C for GAPDH) for 45 s, and host plants of B. thibetaria are mainly conifers belonging to 72 °C for 1 min, and a final 10 min at 72 °C. PCR condi- the gymnosperms (Chen et al. 1982; Zheng & Yang 1991) tions for 16S was as follows: denaturation at 94 °C for from 1000 to 3000 m. However, the precise relationship 3 min, followed by 15 cycles at 94 °C for 30 s, annealing between the three species is unclear. temperature at 55–60 °C for 30 s, 72 °C for 1 min, fol- During our work of phylogeographic analysis of B. pan- lowed by 25 cycles at 94 °C for 35 s, annealing tempera- terinaria, we found that the relationships between B. pan- ture at 50 °C for 30 s, 72 °C for 1 min, and a final 10 min terinaria, B. thibetaria and B. perclara were unusual and that at 72 °C. Multiple alignments were generated using B. panterinaria seemed paraphyletic. However, we were not CLUSTALX (Thompson et al. 1997). Sequences of all pri- sure whether this phenomenon was authentic, because the mers used in this study are listed in Appendix S1. sampling numbers were small. So, in this study, we used Sequences were deposited in GenBank; the accession num- more samples, more data sets and more methods to explore bers are provided in Appendix S2. the relationships of the three species in detail. Nowadays, complete mitochondrial genomes (mitogenomes) can pro- Calculating interspecific sequence divergence and duce more robust and stable phylogenetic reconstructions constructing the NJ tree than those which rely on only part of the mtDNA, and ‘Distance-based method’ and ‘tree-based method’ were have been increasingly applied to a variety of phylogenetic used to ascertain the relationship of the three Biston spe- and phylogeographic studies in invertebrates (Ingman et al. cies. All COI sequences of the three species were used to 2001; Ma et al. 2012; Cameron 2014; Liu et al. 2015; Yang calculate the interspecific sequence divergence and con- et al. 2015). So the phylogenetic relationships of B. panteri- struct the neighbour-join (NJ) tree by MEGA 5.05 (Tamura naria, B. thibetaria and B. perclara were reconstructed based et al. 2011) based on Kimura-2-parameter (K2P) distances. on three data sets, involving mitogenomes, three mitochon- NJ analysis (Saitou & Nei 1987) was used to examine drial DNA (mtDNA) loci and three nuclear DNA whether these three species were effective and authentic. 74 ª 2016 Royal Swedish Academy of Sciences, 46, 1, January 2017, pp 73–84 o C R. Cheng et al. Budding speciation in three Biston species Fig. 1 Sampling sites of B. perclara, B. thibetaria and B. panterinaria. Colours represent different species. Species tree estimation three species to obtain important characters. Morphological A species tree of the three species was constructed using taxonomic methods were used to examine external features Bayesian phylogenetics and phylogeography (BPP v.2.0; and genitalia preparations. The terminology of the genitalia Yang & Rannala 2010) with the full phased data set for the follows that of Pierce (1914, reprint 1976), Klots (1970) and three mitochondrial loci.